Affinity-Resolved Size Exclusion Chromatography Coupled to Mass Spectrometry: A Novel Tool to Study the Attribute-and-Function Relationship in Therapeutic Monoclonal Antibodies.

Assessment of critical quality attributes (CQAs) is an important aspect during the development of therapeutic monoclonal antibodies (mAbs). Attributes that affect either the target binding or Fc receptor engagement may have direct impacts on the drug safety and efficacy and thus are considered as CQAs. Native size exclusion chromatography (SEC)-based competitive binding assay has recently been reported and demonstrated significant benefits compared to conventional approaches for CQA identification, owing to its faster turn-around and higher multiplexity. Expanding on the similar concept, we report the development of a novel affinity-resolved size exclusion chromatography-mass spectrometry (AR-SEC-MS) method for rapid CQA evaluation in therapeutic mAbs. This method features wide applicability, fast turn-around, high multiplexity, and easy implementation. Using the well-studied Fc gamma receptor III-A (FcγRIIIa) and Fc interaction as a model system, the effectiveness of this method in studying the attribute-and-function relationship was demonstrated. Further, two case studies were detailed to showcase the application of this method in assessing CQAs related to antibody target binding, which included unusual N-linked glycosylation in a bispecific antibody and Met oxidation in a monospecific antibody, both occurring within the complementarity-determining regions (CDRs).

An Online Native Mass Spectrometry Approach for Fast, Sensitive, and Quantitative Assessment of Adeno-Associated Virus Capsid Content Ratios.

Adeno-associated viruses (AAVs) have emerged as a leading platform for in vivo therapeutic gene delivery and offer tremendous potential in the treatment and prevention of human disease. The fast-paced development of this growing class of therapeutics, coupled with their intrinsic structural complexity, places a high demand on analytical methods capable of efficiently monitoring product quality to ensure safety and efficacy, as well as to support manufacturing and process optimization. Importantly, the presence and relative abundance of both empty and partially filled AAV capsid subpopulations are of principal concern, as these represent the most common product-related impurities in AAV manufacturing and have a direct impact on therapeutic potential. For this reason, the capsid content, or ratio of empty and partial capsids to those packaged with the full-length therapeutic genome, has been identified by regulatory agencies as a critical quality attribute (CQA) that must be carefully controlled to meet clinical specifications. Established analytical methods for the quantitation of capsid content ratios often suffer from long turnaround times, low throughput, and high sample demands that are not well-suited to the narrow timelines and limited sample availability typical of process development. In this study, we present an integrated online native mass spectrometry platform that aims to minimize sample handling and maximize throughput and robustness for rapid and sensitive quantitation of AAV capsid content ratios. The primary advantages of this platform for AAV analysis include the ability to perform online buffer exchange under low flow conditions to maintain sample stability with minimal sample dilution, as well as the ability to achieve online charge reduction via dopant-modified desolvation gas. By exploiting the latter, enhanced spectral resolution of signals arising from empty, partial, and full AAV capsids was accomplished in the m/z domain to facilitate improved spectral interpretation and quantitation that correlated well with the industry standard analytical ultracentrifugation (AUC) method for capsid content ratio determination. The utility of this approach was further demonstrated in several applications, including the rapid and universal screening of different AAV serotypes, evaluation of capsid content for in-process samples, and the monitoring of capsid stability when subjected to thermal stress conditions.

Enrichment strategy and initial characterization of heterodimers enriched from a co-formulated cocktail of therapeutic antibodies against SARS-COV-2.

Co-formulation of multiple drug products is an efficient and convenient approach to simultaneously deliver multiple biotherapeutics with the potentially added benefit of a synergistic therapeutic effect. However, co-formulation also increases the risk of heteromeric interactions, giving rise to unique impurities with unknown efficacy and immunogenicity. Therefore, it is critical to develop methods to evaluate the risk of heteromers as an impurity that could affect potency, efficacy, and/or immunogenicity. The most direct strategy to evaluate antibody heteromers is via specific enrichment. However, the fact that antibody heterodimers generated from the co-formulated cocktail share highly similar molar mass and size properties as homodimers natively present in each individual antibody drug product poses a unique purification challenge. Here, we report the path to successful enrichment of heterodimers from co-formulated REGEN-COVⓇ and discuss its potential impacts on drug quality.

Discovery and Characterization of an Acid-Labile Serine-Lysine Cross-Link in Antibody High-Molecular-Weight Species Using a Multipronged Mass Spectrometry Approach.

Characterizing the cross-links responsible for the covalent high-molecular-weight (HMW) species in therapeutic monoclonal antibodies (mAbs) is of great importance as it not only provides a framework for risk assessment but also offers insights for process improvement. However, owing to the complexity and low abundance, identification of novel and unknown cross-links in mAb products can be very challenging. Here, applying a multipronged MS-based approach, we report the discovery of a novel covalent cross-link formed via an imine bond between lysine and serine residues. In particular, this Ser-Lys cross-link was found to be acid-labile and can be easily overlooked by conventional LC-MS techniques operated at low pH. It is worth noting that although imine-based cross-link has been previously reported in collagen protein cross-linking, this is the first time that a Ser-Lys cross-link has been found in a mAb product that contributes to covalent HMW species formation.

A generic platform to couple affinity chromatography with native mass spectrometry for the analysis of therapeutic monoclonal antibodies.

Affinity chromatography coupled with native mass spectrometry has emerged as a powerful tool for the analysis of therapeutic monoclonal antibodies (mAbs). Exploiting the specific interactions between mAbs and their ligands, these methods not only provide orthogonal means to study the highly complex mAb attributes, but also offer insights on their biological relevance. Despite the great promise, application of affinity chromatography - native mass spectrometry in routine mAb characterization has been limited, largely due to the complicated experimental set up. In this study, we introduced a generic platform to facilitate the online coupling of different affinity separation modes with native mass spectrometry. Built upon a recently introduced native LC-MS platform, this new strategy can accommodate a wide range of chromatographic conditions, and therefore, allow greatly simplified experimental set up and facile swapping of affinity separation modes. The utility of this platform was demonstrated by successful online coupling of three affinity chromatography methods (protein A, FcγRIIIa, and FcRn) with native mass spectrometry. The developed protein A-MS method was tested both in a "bind-and-elute" mode for rapid mAb screening and in a high-resolution resolving mode to study mAb species with altered protein A affinity. The FcγRIIIa-MS method was applied to achieve glycoform-resolved analyses of both IgG1 and IgG4 subclass molecules. The FcRn-MS method was demonstrated in two case studies, where specific post-translational modifications and Fc mutations were known to alter FcRn affinities.

A competitive binding-mass spectrometry strategy for high-throughput evaluation of potential critical quality attributes of therapeutic monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs) have a propensity to host a large number of chemical and enzymatical modifications that need to be properly assessed for their potential impact on target binding. Traditional strategies of assessing the criticality of these attributes often involve a laborious and low-throughput variant enrichment step prior to binding affinity measurement. Here, we developed a novel competitive binding-based enrichment strategy followed by mass spectrometry analysis (namely, competitive binding-MS) to achieve high-throughput evaluation of potential critical quality attributes in therapeutic mAbs. Leveraging the differences in target binding capability under competitive binding conditions, the criticality of multiple mAb attributes can be simultaneously evaluated by quantitative mass spectrometry analysis. The utility of this new workflow was demonstrated in three mAb case studies, where different post-translational modifications occurring within the complementarity-determining regions were successfully interrogated for their impact on antigen binding. As this workflow does not require prior enrichment (e.g., by forced degradation or liquid chromatography fractionation) of the variants, it is particularly valuable during the mAb candidate developability assessment, where fast turn-around time is highly desired to assist candidate selection.Abbreviations: ACN: acetonitrile; ADCC: antibody-dependent cell-mediated cytotoxicity; AEX: anion exchange chromatography; bsAb: bispecific antibody; CDC: complement-dependent cytotoxicity; CDR: complementarity-determining region; CML: carboxymethylation; CQA: critical quality attribute; DDA: data-dependent acquisition; DMSO: dimethyl sulfoxide; DTT: dithiothreitol; FA: formic acid; Fab: Fragment antigen-binding; FcRn: neonatal Fc receptor; HC: heavy chain; HIC: hydrophobic interaction chromatography; IAA: iodoacetamide; IEX: ion exchange chromatography; LC: light chain; mAb monoclonal antibody; msAb: monospecific antibody; MS: mass spectrometry; PBS: phosphate-buffered saline; pI: isoelectric point; PTM: post-translational modification; SCX: strong cation exchange chromatography; SEC: size exclusion chromatography; SPR: surface plasmon resonance; XIC: extracted ion chromatography.

Coupling Anion Exchange Chromatography with Native Mass Spectrometry for Charge Heterogeneity Characterization of Monoclonal Antibodies.

Despite the recent success of coupling anion exchange chromatography with native mass spectrometry (AEX-MS) to study anionic proteins, the utility of AEX-MS methods in therapeutic monoclonal antibody (mAb) characterization has been limited. In this work, we developed and optimized a salt gradient-based AEX-MS method and explored its utility in charge variant analysis of therapeutic mAbs. We demonstrated that, although the developed AEX-MS method is less useful for IgG1 molecules that have higher isoelectric points (pIs), it is an attractive alternative for charge variant analysis of IgG4 molecules. By elevating the column temperature and lowering the mAb pI through PNGase F-mediated deglycosylation, the chromatographical resolution from AEX separation can be significantly improved. We also demonstrated that, after PNGase F and IdeS digestion, the AEX-MS method exhibited excellent resolving power for multiple attributes in the IgG4 Fc region, including unprocessed C-terminal Lys, N-glycosylation occupancy, and several conserved Fc deamidations, making it ideally suited for multiple attribute monitoring (MAM). Through fractionation and peptide mapping analysis, we also demonstrated that the developed AEX-MS method can provide site-specific and isoform-resolved separation of Fc deamidation products, allowing rapid and artifact-free quantitation of these modifications without performing bottom-up analysis.

Post-Column Denaturation-Assisted Native Size-Exclusion Chromatography-Mass Spectrometry for Rapid and In-Depth Characterization of High Molecular Weight Variants in Therapeutic Monoclonal Antibodies.

The high molecular weight (HMW) size variants present in therapeutic monoclonal antibody (mAb) samples need to be closely monitored and characterized due to their impact on product safety and efficacy. Because of the complexity and often low abundances in final drug substance (DS) samples, characterization of such HMW species is challenging and traditionally requires offline enrichment of the HMW species followed by analysis using various analytical tools. Here, we report the development of a postcolumn denaturation-assisted native SEC-MS method that allows rapid and in-depth characterization of mAb HMW species directly from unfractionated DS samples. This method not only provides high-confidence identification of HMW complexes based on accurate mass measurement of both the intact assembly and the constituent subunits but also allows in-depth analysis of the interaction nature and location. In addition, using the extracted ion chromatograms, derived from high-quality, native-like mass spectra, the elution profiles of each noncovalent and/or nondissociable complex can be readily reconstructed, facilitating the comprehension of a complex HMW profile. The utility of this novel method was demonstrated in different applications, ranging from enriched HMW characterization at late stage development, comparability assessment due to process changes, and forced degradation study of coformulated mAbs. As this method does not require prior enrichment, it is thus desirable for providing both rapid and in-depth characterization of HMW species during the development of therapeutic mAbs.

Development of a chromatography-free method for high-throughput MS-based bioanalysis of therapeutic monoclonal antibodies.

Aim: Our objective was to test the feasibility of developing an LC-free, MS-based approach for high-throughput bioanalysis of humanized therapeutic monoclonal antibodies. Methodology: A universal tryptic peptide from human IgG1, IgG3 and IgG4 was selected as the surrogate peptide for quantitation. After tryptic digestion, the surrogate peptide was fractionated via solid-phase extraction before being subjected to direct infusion-based MS/MS analysis. A high-resolution, multiplexed (MSX = 2) parallel reaction monitoring method was developed for data acquisition. Results & conclusion: This proof-of-concept study demonstrated the feasibility of achieving high-throughput MS-based bioanalysis of monoclonal antibodies using an LC-free workflow with sensitivity comparable to conventional LC-MS/MS-based methods.

Versatile, Sensitive, and Robust Native LC-MS Platform for Intact Mass Analysis of Protein Drugs.

Over the past several years, hyphenation of native (nondenaturing) liquid chromatography (nLC) methods, such as size exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophobic interaction chromatography (HIC) with mass spectrometry (MS) have become increasingly popular to study the size, charge, and structural heterogeneity of protein drug products. Despite the availability of a wide variety of nLC-MS methods, an integrated platform that can accommodate different applications is still lacking. In this study, we described the development of a versatile, sensitive, and robust nLC-MS platform that can support various nLC-MS applications. In particular, the developed platform can tolerate a wide range of LC flow rates and high salt concentrations, which are critical for accommodating different nLC methods. In addition, a dopant-modified desolvation gas can be readily applied on this platform to achieve online charge-reduction native MS, which improves the characterization of both heterogeneous and labile biomolecules. Finally, we demonstrated that this nLC-MS platform is highly sensitive and robust and can be routinely applied in protein drug characterization.

Characterization of Adeno-Associated Virus Capsid Proteins Using Hydrophilic Interaction Chromatography Coupled with Mass Spectrometry.

To support adeno-associated virus (AAV)-based gene therapy development, characterization of the three capsid viral proteins (VP; VP1/VP2/VP3) from recombinant AAV can offer insights on capsid identity, heterogeneity, and product and process consistency. Intact protein mass analysis is a rapid, reliable, and sensitive method to confirm AAV serotypes based on accurate mass measurement of the constituent capsid proteins. Compared to commonly applied reversed-phase liquid chromatography (RPLC) methods, we demonstrated that, using a wide-pore amide-bonded column, hydrophilic interaction chromatography (HILIC) could achieve improved separation of VPs from a variety of AAV serotypes using a generic method prior to MS detection. Moreover, HILIC-based separation was shown to be particularly sensitive in detecting capsid protein variants resulting from different post-translational modifications (PTMs) (e.g. phosphorylation and oxidation) and protein backbone clippings, making it ideally suited for capsid heterogeneity characterization. To overcome the challenges associated with low protein concentrations of AAV samples, as well as the trifluoroacetic acid (TFA)-induced ion suppression during HILIC-MS analysis, different strategies were implemented to improve method sensitivity, including increasing the HILIC column loading and the application of a desolvation gas modification device. Finally, we demonstrated that this integrated HILIC-FLR-MS method can be generically applied to characterize a variety of AAV serotype samples at low concentrations without any sample treatment to achieve unambiguous serotype identification, stoichiometry assessment, and PTM characterization.

An 18O-Labeling Assisted LC-MS Method for Accurate Quantitation of Unprocessed C-Terminal Lysine in Therapeutic Monoclonal Antibodies.

Unprocessed C-terminal lysine (C-term Lys) is one of the most common causes for the formation of basic variants in therapeutic monoclonal antibodies (mAbs). Although the C-term Lys variants are routinely quantified by a LC-MS-based peptide mapping method using the relative MS responses from both C-terminal peptides (with and without Lys), this approach often leads to overestimation of Lys-containing peptide due to the intrinsic difference in ionization efficiency. Herein, we report an 18O-labeling assisted LC-MS method, which takes advantage of the carboxypeptidase B-catalyzed Lys removal and 18O-labeling to achieve improved accuracy of C-term Lys quantitation. The fidelity of this method was first demonstrated using synthetic peptide mixture standards that mimic a wide range of C-term Lys levels. Finally, the newly developed method was applied in a case study where C-term Lys variants in mAb samples manufactured from different processes were accurately quantified and compared. This new method provides a valuable solution for studies where accurate C-term Lys levels are needed to assist decision-making and root-cause investigation.

Online coupling of analytical hydrophobic interaction chromatography with native mass spectrometry for the characterization of monoclonal antibodies and related products.

Native mass spectrometry (native MS) has seen tremendous development and an increase in application over the past decade for the study of proteins and protein complexes. Although conventionally performed using a static nanospray emitter in an offline fashion, native MS has been increasingly applied in hyphenated methods, where a wide variety of separation techniques are directly coupled to online native MS detection. Those new developments have greatly expanded the utility of native MS in protein biopharmaceutical characterization. Analytical hydrophobic interaction chromatography (HIC) method, although frequently used for the characterization of monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs), has rarely been explored for online coupling with native MS. This is largely due to the high salt concentrations used in HIC analysis that are not compatible with direct MS detection. In this study, we overcame this challenge via an innovative makeup and splitting flow design and successfully achieved online coupling of analytical HIC separation with native MS detection. The development and experimental setup of this HIC-MS method is outlined in detail to elucidate how this design could tackle the high salt concentrations used in HIC separation and ultimately achieve both good chromatographic resolution and MS data quality. Subsequently, the utility of this HIC-MS method was demonstrated in three different applications, where a mAb mixture, mAb molecular variants resulting from PTMs, and a Cys-based ADC mimic were all readily characterized in detail. Unlike previously reported HIC-MS methods, this newly developed method utilizes an analytical scale HIC column with conventional ligand so that the achieved separation profile is highly comparable to those obtained by a standard HIC-UV method. As a result, this HIC-MS method not only provides an alternative approach for in-depth characterization of mAbs and related products during their development but could also be readily applied to assist peak assignment and identity elucidation for the HIC-UV method used in quality control.

Coupling Mixed-Mode Size Exclusion Chromatography with Native Mass Spectrometry for Sensitive Detection and Quantitation of Homodimer Impurities in Bispecific IgG.

Detection and quantitation of homodimer impurities in therapeutic bispecific antibody (bsAb) drug products is essential to support development and quality control (QC) release. LC-MS-based techniques have been frequently applied for this analysis. However, sensitive detection of low-abundance homodimer impurities can still be challenging for regular workflows, which is largely due to the lack of chromatographic resolution between the impurities and the main bsAb species. Here, we report the development of a novel analytical method, which couples mixed-mode size exclusion chromatography (mmSEC) with online native MS detection (mmSEC-MS) for highly sensitive detection and quantitation of homodimer impurities in bsAb samples. Secondary interactions between the protein analytes and the column matrix, which are typically unwanted in SEC applications, are utilized to separate mAb species with similar hydrodynamic volume but different surface characteristics. Using four different bsAbs as testing standards, we demonstrated the versatility of this method in separating homodimer species from bsAb based on either electrostatic interaction or hydrophobic interaction, which was easily achieved by utilizing SEC columns with different properties as well as modulating the salt concentrations. The chromatographic separation between homodimer impurities and bsAb, as achieved by the mmSEC method, was demonstrated to be critical for the improved sensitivity in detecting low-abundance homodimer impurities (LOD from 0.01% to 0.1%). To the best of our knowledge, this newly developed mmSEC-MS method represents the most sensitive MS-based technique in both detection and quantitation of homodimer impurities in bsAb samples.

Genetic Differentiation of Eastern Honey Bee (Apis cerana) Populations Across Qinghai-Tibet Plateau-Valley Landforms.

Many species of high-altitude plateaus tend to be narrowly distributed along river valleys at lower elevations due to a limitation of suitable habitats. The eastern honeybee (Apis cerana) is such a species and this study explored the effects of long and narrow geographic distributions on honeybee populations. Genetic differentiation and diversity were assessed across populations of the southeastern Qinghai-Tibet Plateau. A total of 492 honeybee samples from eight sampling sites in four valleys were analyzed for the genetic differentiation and diversity of 31 microsatellite loci and mitochondrial tRNAleu-COII fragments. The following results were obtained: (1) Microsatellite genetic differentiation coefficients (F ST) ranged from 0.06 to 0.16, and mitochondrial F ST estimates ranged from 0.18 to 0.70 for different sampling sites in the same valley, indicating genetic differentiation. (2) Honeybees in adjacent valleys were also genetically differentiated. The F ST of microsatellites and mitochondria were 0.04-0.29 and 0.06-0.76, respectively. (3) Likely a result of small population sizes, the observed genetic diversity was low. The observed impedance of honeybee gene flow among valleys increased both genetic differentiation and population numbers in the Qinghai-Tibet Plateau. This study contributes significantly to the current understanding of the mechanism underlying population genetic differentiation and highlights the potential effects of utilizing genetic resources that are subject to the ecological conditions of the long and narrow geographic distributions of plateau-valley landforms.

Simple Approach for Improved LC-MS Analysis of Protein Biopharmaceuticals via Modification of Desolvation Gas.

LC-MS based analysis of protein biopharmaceuticals could benefit from improved data quality, which can subsequently lead to improved drug characterization with higher confidence and less ambiguity. In this study, we created a simple device to modify the desolvation gas on a Q-Exactive mass spectrometer and to demonstrate the utility in improving both peptide mapping analysis and intact mass analysis, the two most routinely and widely applied LC-MS techniques in protein biopharmaceutical characterization. By modifying the desolvation gas with acid vapor from propionic acid (PA) and isopropanol (IPA), the ion suppression effects from trifluoroacetic acid (TFA) in a typical peptide mapping method can be effectively mitigated, thus leading to improved MS sensitivity. By modifying the desolvation gas with base vapor from triethylamine (TEA), the charge reduction effect can be achieved and utilized to improve the spectral quality from intact mass analysis of protein biopharmaceuticals. The approach and device described in this work suggests a low-cost and practical solution to improve the LC-MS characterization of protein biopharmaceuticals, which has the potential to be widely implemented in biopharmaceutical analytical laboratories.

Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry.

In therapeutic monoclonal antibody (mAb) development, charge heterogeneity of a mAb molecule is often associated with critical quality attributes and is therefore monitored throughout development and during QC release to ensure product and process consistency. Elucidating the cause of each charge variant species is an involved process that often requires offline fractionation by ion exchange chromatography (IEX) followed by mass spectrometry (MS) analysis, largely due to the incompatibility of conventional IEX buffers for direct MS detection. In this study, we have developed a method that combines a generic strong cation exchange (SCX) chromatography step with ultrasensitive online native MS analysis (SCX-MS) optimized for mAb separation and detection. As demonstrated by analyzing mAb molecules with a wide range of pI (isoelectric point) values, the developed method can consistently achieve both high-resolution IEX separation and ultrasensitive MS detection of low-abundance charge variant species. Using this method, we analyzed the charge heterogeneity of NISTmAb reference material 8671 (NISTmAb) at both whole antibody and subdomain levels. In particular, due to the high sensitivity, a nonconsensus Fab glycosylation site, present at a very low level (<0.1%), was directly detected in the NISTmAb sample without any enrichment. The structure and location of this Fab glycosylation was further characterized by peptide mapping analysis. Despite the extensive characterization of NISTmAb material in previous studies, this is the first time that this Fab-glycosylated variant has been identified in the NISTmAb, demonstrating the value of this new method in achieving a more comprehensive characterization of charge heterogeneity for therapeutic mAbs.

Characterization of product-related low molecular weight impurities in therapeutic monoclonal antibodies using hydrophilic interaction chromatography coupled with mass spectrometry.

Traditional SDS-PAGE method and its modern equivalent CE-SDS method are both widely applied to assess the purity of therapeutic monoclonal antibody (mAb) drug products. However, structural identification of low molecular weight (LMW) impurities using those methods has been challenging and largely based on empirical knowledges. In this paper, we present that hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry analysis is a novel and orthogonal method to characterize such LMW impurities present within a purified mAb drug product sample. We show here that after removal of N-linked glycans, the HILIC method separates mAb-related LMW impurities with a size-based elution order. The subsequent mass measurement from a high-resolution accurate mass spectrometer provides direct and unambiguous identification of a variety of low-abundance LMW impurities within a single LC-MS analysis. Free light chain, half antibody, H2L species (antibody possessing a single light chain) and protein backbone-truncated species can all be confidently identified and elucidated in great detail, including the truncation sites and associated post-translational modifications. It is worth noting that this study provides the first example where the H2L species can be directly detected in a mAb drug product sample by intact mass analysis without prior enrichment.

Evaluation of Nonferrous Metals as Potential In Vivo Tracers of Transferrin-Based Therapeutics.

Transferrin (Tf) is a promising candidate for targeted drug delivery. While development of such products is impossible without the ability to monitor biodistribution of Tf-drug conjugates in tissues and reliable measurements of their levels in blood and other biological fluids, the presence of very abundant endogenous Tf presents a significant impediment to such efforts. Several noncognate metals have been evaluated in this work as possible tracers of exogenous transferrin in complex biological matrices using inductively coupled plasma mass spectrometry (ICP MS) as a detection tool. Placing Ni(II) on a His-tag of recombinant Tf resulted in formation of a marginally stable protein-metal complex, which readily transfers the metal to ubiquitous physiological scavengers, such as serum albumin. An alternative strategy targeted iron-binding pockets of Tf, where cognate Fe(III) was replaced by metal ions known to bind this protein. Both Ga(III) and In(III) were evaluated, with the latter being vastly superior as a tracer (stronger binding to Tf unaffected by the presence of metal scavengers and the retained ability to associate with Tf receptor). Spiking serum with indium-loaded Tf followed by ICP MS detection demonstrated that protein quantities as low as 0.04 nM can be readily detected in animal blood. Combining laser ablation with ICP MS detection allows distribution of exogenous Tf to be mapped within animal tissue cross-sections with spatial resolution exceeding 100 µm. The method can be readily extended to a range of other therapeutics where metalloproteins are used as either carriers or payloads. Graphical Abstract ᅟ.

A new liquid chromatography-mass spectrometry-based method to quantitate exogenous recombinant transferrin in cerebrospinal fluid: a potential approach for pharmacokinetic studies of transferrin-based therapeutics in the central nervous systems.

Transferrin (Tf) is an 80 kDa iron-binding protein that is viewed as a promising drug carrier to target the central nervous system as a result of its ability to penetrate the blood-brain barrier. Among the many challenges during the development of Tf-based therapeutics, the sensitive and accurate quantitation of the administered Tf in cerebrospinal fluid (CSF) remains particularly difficult because of the presence of abundant endogenous Tf. Herein, we describe the development of a new liquid chromatography-mass spectrometry-based method for the sensitive and accurate quantitation of exogenous recombinant human Tf in rat CSF. By taking advantage of a His-tag present in recombinant Tf and applying Ni affinity purification, the exogenous human serum Tf can be greatly enriched from rat CSF, despite the presence of the abundant endogenous protein. Additionally, we applied a newly developed (18)O-labeling technique that can generate internal standards at the protein level, which greatly improved the accuracy and robustness of quantitation. The developed method was investigated for linearity, accuracy, precision, and lower limit of quantitation, all of which met the commonly accepted criteria for bioanalytical method validation.